Amitriptyline is a tricyclic antidepressant widely used in the treatment of chronic pain. The objective of the present study was to investigate the potential cytotoxic effects of amitriptyline in human fibroblasts primary culture. Human fibroblast cells were cultured from healthy subjects and incubated with 50 μM and 100 μM amitriptyline. Cell counting was performed to study dose-dependency of toxicity. Lipid peroxidation analysis and western blotting for antioxidants catalase and mitochondrial superoxide dismutase (MnSOD) were carried out in order to evaluate oxidative stress. To investigate mitochondria damage the following determinations were made: cytochrome c, citrate synthase, and mitochondrial membrane potential (ΔΨm). Amitriptyline reduced significantly the number of cultured cells, resulting in a decrease of 45.2%, 65.0% and 94.9% when treated with 20 μM, 50 μM and 100 μM amitriptyline, respectively. This drug enhanced the production of oxidized products during lipid peroxidation, inverting the reduced/oxidized ratio to 25% reduction and 75% oxidation after 24 h of amitriptyline administration. A decreased in catalase protein levels has been also observed. Moreover, amitriptyline treatment induced a significant decrease of cytochrome c, ΔΨm, and citrate synthase activity; revealing mitochondrial damage. These findings suggest that amitriptyline has a strong cytotoxic effect in human fibroblasts, decreasing growth rate and mitochondrial activity, and increasing oxidative stress.